The body's pathogen detection system is critical to host defense - initiating inflammation and tailoring the immune response. Pattern recognition receptors (PRRs) bind pathogen-associated molecules and activate a network of transcription factors (TFs) that regulate host defense genes. The TFs IRF3, IRF5 and IRF7 are central to PRR signaling in response to viruses and intracellular pathogens. Despite common DNA binding specificities and activation patterns, IRF3, IRF5 and IRF7 (hereafter IRF3/5/7) perform overlapping yet distinct roles in host defense. Much is known regarding differences in the signaling events upstream of IRF3/5/7 activation; however, little is known about how IRF3/5/7 discriminate their individual target genes at the DNA level to elicit different biological outcomes and tailor the immune response to pathogens. This proposal aims to capitalize upon advances in genome-scale techniques and datasets to address the issue of IRF3/5/7 cis-regulatory specificity in the gene regulatory network governing pathogen detection. Protein binding microarrays (PBMs) will be used to characterize the DNA binding of IRF3/5/7 dimers, addressing deficiencies in our current models. To examine how IRF3/5/7 binding differences contribute to regulatory differences, PBM binding data will be integrated with chromatin immunoprecipitation (ChIP) and gene expression datasets. Hypotheses will be tested using cell-based reporter assays (Aim 1). To examine the role of NF-?B - a known cofactor of IRF3/5/7 - we will (1) use ChIP-seq to examine IRF binding in wild- type and NF-?B knockdown cells, and (2) use PBMs to examine DNA binding specificity of IRF- NF-?B complexes (Aim 2). We hypothesize that differential interactions between the IRFs and NF-?B will contribute to their individual biological roles. To uncover novel IRF cofactors that might contribute to regulatory differences, we will use combined computational and proteomics approaches. Computational motif analysis will be applied to ChIP-seq datasets to identify motifs (and cognate factors) associated with IRF binding. Traditional separation techniques coupled with mass spectrometry (mass-spec) will be used to identify novel factors from cell lysates that co-bind with IRF factors on DNA oligonucleotides (Aim 3). These studies are expected to provide critical insights into the mechanisms underlying different IRF3/5/7 functions. Delineating the role and scope of cofactor interactions in IRF3/5/7 function will provide insight into the interdependence of signaling pathways in the host defense response, and may reveal new opportunities for therapeutic intervention in inflammation. More broadly, this proposal aims to clarify PRR-IRF3/5/7 signaling specificity with a view to a more complete model of host defense that can address phenomena such as antagonism between infecting pathogens, and the relation between infection and autoimmune diseases such as systemic lupus erythematosus (SLE), which is strongly associated with altered PRR-IRF5/7 signaling.